An important problem in gene therapy is the control of the transcription and translation of an effector gene which is inserted into the cell. At the level of transcription, this control is made possible by adding on a promoter or enhancer sequence upstream of the coding sequence of the effector gene. The "promoter sequence" is understood as being a gene segment to which regulatory proteins, the so-called transcription factors, which in their totality activate the transcription of the downstream effector gene, are able to bind. Those regions which lie in the direction of transcription are designated "downstream" sequences, whereas sequences which are arranged in the opposite direction are designated "upstream" sequences. An "effector gene" is generally understood as being a structural gene whose gene product has, for example, a desirable effect in the gene therapy sense.
Such promoter or enhancer sequences can be non-cell-specific, cell-specific, virus-specific, metabolism specific or cell cycle-specific. Examples of these promoter sequences and their use, e.g. for the gene therapy of different diseases, are listed in Patent Applications WO 96/06940, WO 96/06938, WO 96/06941 and WO 96/06939. In addition, these patent applications present techniques and examples for combining these promoter sequences, e.g. for the purpose of controlling an effector gene cell-specifically and cell cycle-specifically.
Depending on the choice and combinations of the promoters, these promoters bring about a more or less restricted and/or more or less powerful transcription of the effector gene.
The endothelial cell is an example of an advantageous target cell for gene therapy, on the one hand because endothelial cells are directly accessible to gene constructs which are injected into the circulatory system and, on the other hand, because they are directly involved in the development and progress of a number of disorders, such as tumor diseases, inflammations, allergies, autoimmune diseases, organ rejection reactions and circulatory and coagulation disorders, and also in healing processes, and/or are directly adjacent to the site of these disorders.
As a rule, target cell-specific promoters are promoters of genes for those proteins which are formed particularly vigorously, or to a large extent exclusively, in the relevant target cell. In the case of the endothelial cell, endoglin is an example of one of these proteins.
Endoglin is a non-signal-transferring receptor of TGFP (Gougos et al., J. Biol. Chem. 265, 8361 (1990), Cheifetz, J. Biol. Chem. 267, 19027 (1992), Moren et al., BBRC 189, 356 (1992)). While it occurs in small quantities on normal endothelium, it is expressed to an increased extent on proliferating endothelium (Westphal et al., J. Invest. Derm. 100, 27 (1993), Burrows et al., Pharmac. Ther. 65, 155 (1994). No further information is available with regard to promoter strength and cell specificity. Despite the fact that the endoglin gene has been known for about 4 years (Bellon et al., (1993), it has not so far been possible to isolate the endoglin promoter.
The cDNA sequence for human endoglin has been described by Bellon et al. (Eur. J., Immunol. 23, 2340 (1993)), while that for murine endoglin has been described by Ge et al. (Gene 138, 201 (1994)). While sequence information is available for a part of the 5'-non-translated region of the endoglin gene, nothing is known about the function of this region or about the promoter region.
The VEGF receptor is another endothelial cell-specific protein. In this case, two receptors are distinguished (Plate et al., Int. J. Cancer 59, 520 (1994)): on the one hand, VEGF receptor 1 (flt-1), (de Vries et al., Science 255, 989 (1992)), which contains an fms-like tyrosine kinase in the cytoplasmic moiety, and VEGF receptor 2 (flk-1, KDR), (Terman et al., BBRC 187, 1579 (1992)), which contains a tyrosine kinase in the cytoplasmic moiety. Both receptors are found almost exclusively on endothelial cells (Senger et al., Cancer Metast. Rev. 12, 303 (1993)).
Other endothelial cell-specific receptor tyrosine kinases are tie-1 or tie-2 (Partanen et al., Mol. Cell. Biol. 12, 1698 (1992), Schnurch und Risau, Development 119, 957 (1993), Dumont et al., Oncogene 7, 1471 (1992)), and the B61 receptor (Eck receptor), (Bartley et al., Nature 368, 558 (1994), Pandey et al., Science 268, 567 (1995), van der Geer et al., Ann. Rev. Cell. Biol. 10, 251 (1994)).
Other endothelial cell-specific proteins are the B61 molecule, which represents the ligand for the B61 receptor (Holzman et al., J. Am. Soc. Nephrol. 4, 466 (1993), Bartley et al., Nature 368, 558 (1994)), endothelin, in particular endothelin B (O'Reilly et al., J. Cardiovasc. Pharm. 22, 18 (1993), Benafti et al., J. Clin Invest 91, 1149 (1993), O'Reilly et al., BBRC 193, 834 (1993)), whose promoter sequence has been described by Benafti et al., J. Clin. Invest. 91, 1149 (1993), endothelin 1 (Yanasigawa et al., Nature 332, 411 (1988)), whose promoter sequence has been described by Wilson et al., Mol. Cell. Biol. 10, 4654 (1990), endothelin receptors, in particular the endothelin B receptor (Webb et al., Mol. Pharmacol. 47, 730 (1995), Haendler et al. J. Cardiovasc. Pharm. 20, 1 (1992)), mannose 6-phosphate receptors (Perales et al., Eur. J. Biochem. 226, 225 (1994)), whose promoter sequences have been described by Ludwig et al. (Gene 142, 311 (1994), Oshima et al., (J. Biol. Chem. 263, 2553 (1988)) and Pohlmann et al. (PNAS USA 84, 5575 (1987)), and von Willebrand factor (vWF), whose promoter sequence has been described by Jahroudi and Lynch (Mol. Cell. Biol. 14, 999 (1994)), Ferreira et al. (Biochem. J. 293, 641 (1993)) and Aird et al. (PNAS USA 92, 4567 (1995)).
Other endothelial cell-specific proteins are IL-1 in the form, for example, of IL-1(x and IL-1p, which are produced by activated endothelial cells (Warner et al., J. Immunol. 139, 1911 (1987)) and whose promoter sequences have been described by Hangen et al., Mol. Carcinog. 2, 68 (1986), Turner et al., J. Immunol. 143, 3556 (1989), Fenton et al., J. Immunol. 138, 3972 (1 987), Bensi et al., Cell Growth Diff. 1, 491 (1990), Hiscoft et al., Mol. Cell. Biol. 13, 6231 (1993) and Mori et al., Blood 84, 1688 (1994), IL-1 receptor, whose promoter sequence has been described by Ye et al., PNAS USA 90, 2295 (1993), and vascular cell adhesion molecule (VCAM-1), with the expression of VCAM-1 in endothelial cells being activated by lipopolysaccharides, TNF-(X (Neish et al., Mol. Cell. Biol. 15, 2558 /1995)), IL-4 (lademarco et al., J. Clin. Invest. 95, 264 (1995)) and IL-5 (Marni et al., J. Clin. Invest. 92, 1866 (1993)). The promoter sequence of VCAM-1 has been described by Neish et al., Mol. Cell. Biol. 15, 2558 (1995), Ahmad et al., J. Biol. Chem. 270, 8976 (1995), Neish et al., J. Exp. Med. 176, 1583 (1992), Lademareo et al., J. Biol.
Chem. 267, 16323 (1992), and Cybuisky et al., PNAS USA 88, 7859 (1991).
Other endothelial cell-specific promoters are synthetic activator sequences, in that synthetic activator sequences, which are composed of oligomerized binding sites for transcription factors which are preferentially or selectively active in endothelial cells, for example the transcription factor GATA-2, whose binding site in the endothelin I gene is 5'-TTATCT-3' (Lee et al., Biol. Chem. 266, 16188 (1991), Dorfmann et al., J. Biol. Chem. 267, 1279 (1992) and Wilson et al., Mol. Cell. Biol. 10, 4854 (1 990)), can also be used as an alternative to natural endothelium-specific promoters, and the brain-specific, endothelial glucose-1 -transporter, in that brain endothelial cells characteristically express this transporter very strongly in order to effect transendothelial transport of D-glucose into the brain (Gerhart et al., J. Neurosci. Res. 22, 464 (1989)). The promoter sequence has been described by Murakami et al. (J. Biol. Chem. 267, 9300 (1992)).
While being fairly specific for endothelial cells, some of these promoters, for example the promoter for the gene for von Willebrand factor or for the gene for VEGF receptor 1 (flk-1), are, however, only of relatively low activity. While the activity of such "weak" promoters can be increased by combining them with a basal promoter (e.g. SV40) or an enhancer, this then usually leads to an accompanying decrease in specificity. Accordingly, strong promoters are needed for high expression of transgenic materials in endothelial cells.